This week was all about venturing into the wild western blots! To quickly go over the basics of western [blotting], I will go over six major steps. There’s actually a lot more steps and little specifics you need to watch out for doing westerns, which makes it so hard to troubleshoot because there are so many areas you can go wrong! However, even though it is more tedious I think the process is more fun than doing flow and more exhausting (it can take up to 2 days sometimes).
1) Sample preparation- collecting proteins + bradford assay
a. To collect only proteins, we need to get them by lysing the cell first!
b.After getting the proteins, we usually use a microplate reader and stick in a plate filled with samples and known standards of protein concentration to compare the unknowns to the known. We do this to find each sample’s protein concentration and dilute it with other reagents to make it all the same. It takes a lot of math and excel sheets. 🙁
2) Gel electrophoresis- separating the proteins by size
Instead of the gel being laid flat as normal, it is put into a tank and the gel runs vertically standing up!
3) Membrane transfer- it is a sandwich!
ingredients: filter papers, membrane (what the proteins are being transferred onto), gel, and more filter paper
the sandwich recipe!
example membrane results after transfer + developing
4) Blocking- use BSA blocking buffer to prevent non-specific binding
As you can see from the diagram, the blocking agent attaches to areas of non-interest leaving only the targets of interest identifiable for the antibody.
5) Primary and Secondary antibody blocking
a. primary- binds to protein directly
b. secondary- a fluorochrome used to track the primary antibody
6) Detection and analysis- scanning and understanding the image
Using the printer like machine (left), we can the scan the membrane and from the light emitted from the fluorochrome, which should look something like the image on the right! From the positions of the band, we can infer many things. Such as what protein it was, the effect something else had on the protein based on the increased appearance up/down the chromosome position etc.
And that’s it! I actually started to do some westerns on antibodies we were interested, but the western image showed up with wayyy too many bands meaning there a lot of non-specific binding. We are currently trying to trouble shoot and are hoping to redo it with better results. Wish me luck !
Hope you had fun conquering the wild west(erns) with me,